Gel body and a method for biological sterilization control

ABSTRACT

The invention relates to a gel body of a delimited physical form preferably containing water and characterized in that it a) contains an encapsulated apathogenic micro-organism having a known and reproducible resistance; b) has a porous structure with the size of the pores defined, said structure preventing the encapsulated micro-organism from diffusing out but allows a nutrient solution adjusted to the micro-organism to diffuse in and metabolites produced to diffuse; c) is thermally stable in various sterilization, pasteurization or cooking processes; d) is mechanically stable when transported through the process equipment for sterilization, pasteurization or cooking; e) is surface sterile; and f) is transparent in order to allow observation of possible growth of the micro-organism when incubated in a nutrient solution. Also, the invention relates to a method for carrying out biological control of a sterilization, pasteurization, or cooking process, the method being characterized in that one or more gel bodies according to claim 1 are added to the product to be sterilized, that the product containing gel bodies is subjected to sterilization, pasteurization, cooking or other heat treatment, that the gel body or bodies are separated from the sterilized product after the sterilization has been completed, that the gel body or gel bodies are incubated in a nutrient solution, and that possible growth of micro-organisms is indicated.

The present invention relates to a product and a method for biologicalcontrol of a process of the type of sterilization, pasteurization orcooking.

Heat treatment of various products by sterilization, pasteurization orcooking for killing various micro-organisms is applied within the foodindustry, breweries and dairies, public health service, sanitary andmedical article industry, drug industry etc. In monitoring such heattreatment processes within the health service and medical articleindustry so called spore slips of paper are used. Such paper slips arecaused to absorb various types of spores, for example, of Bacillussubtilis and of Bacillus stearothermphilus, which are temperature stableup to specific temperature ranges. These spore slips are then placedtogether with non-sterile sanitary articles for sterilization in anautoclave at the temperature of about 120° C. for about 20 minutes,alternatively in a gaseous atmosphere of ethylene oxide, formaldehyde orheated steam. The spore slips are checked with respect to possiblesurviving spores by the slips being put in a nutrient solution providedwith a pH indicator (e.g. BTB-bromothymol blue). A change of colour ofthe nutrient solution after having been incubated for 1-3 days at 37°C., alternatively 55-60° C., will indicate that the autoclave treatmenthas been incomplete.

The use of the spore slips mentioned above is limited to applicationswhere the risk of contaminating the products and the process equipmentis not crucial to the continued function of the same. This means thatsaid method cannot be accepted for sterility control in food production,particularly not for direct process control of e.g. liquid food productswhere the spores from the spore slips would be transferred directly tothe product and infect the same.

The remaining present alternatives of various types of sterility controlare, for example, to registrate continuously the temperature developmentin the process by controlling the temperature in autoclaves and othersterility reactors. The disadvantage of this procedure resides in thefact that it is only possible to measure at random at specified pointsof the equipment, not directly on each particle in, for example a liquidproduct or a product containing pieces.

The present invention relates to a product and a method for a biologicalsterility control directly in various processes of sterilization,pasteurization or cooking without any risk of contaminating the productto be sterilized and the process equipment. More particularly, accordingto one aspect of the invention it relates to a preferablywater-containing gel body, having a discrete or delimited physical form,said body being characterized in that it

a) contains an encapsulated apathogenic micro-organism having a knownand reproducible resistance;

b) has a porous structure with the size of the pores defined, saidstructure preventing the encapsulated micro-organism from diffusing outbut allowing a nutrient solution adjusted to the micro-organism todiffuse in and metabolites produced to diffuse out;

c) is thermally stable in various sterilization processes;

d) is mechanically stable when transported through the process equipmentfor sterilization, pasteurization or cooking;

e) is surface sterile; and

f) is transparent in order to allow observation of possible growth ofthe micro-organism when incubated in a nutrient solution.

The invention also relates to a method of conducting a biologicalcontrol of a sterilization, pasteurization or cooking process, themethod being characterized by the steps that one or more gel bodiesaccording to the invention are added to the product to be sterilized,that the product containing gel bodies is subjected to conventionalsterilization, and that the gel body or bodies are separated from thesterilized product after completion of the sterilization process andincubated in a nutrient solution adjusted to the micro-organism, wherepossible growth of the micro-organism is indicated, for example, by acolour change of the nutrient solution.

The gel body according to the invention can consist of any suitable gelmaterial having the properties defined under a) to f). For instance, thegel material can consist of polyacrylamide, calcium alginate,polyurethane, gelatine, agar or a combination thereof.

The gel body can consist of one single gel material, e.g.polyacrylamide, preferably, however, it comprises a core of a gelmaterial, e.g. polyacrylamide or gelatine, covered by one or more layersof another gel material, e.g. calcium alginate, with the purpose ofensuring that the gel body be tightly sealed with respect to microorganisms within the temperature range contemplated, thus to avoidinfection of the product to be sterilized.

Gels of polyacrylamide are mechanically strong, thermally stable, easyto handle, and they are completely transparent. They are produced bypolymerization of monomeric acrylamide with a cross-linked copolymer,N,N'-methylenebis-acrylamide (known as "bis") in the presence of freeradicals, which are supplied by a chemical initiator, such as ammoniumpersulphate, or a photochemical initiator, such as riboflavin. Thereaction is usually controlled by addition of TEMED(N,N,N',N'-tetramethylethylenediamine), which furnishes tertiary amines.The pore size of the gel can be varied by a suitable choice of totalacrylamide concentration and the degree of cross-linking. Thetemperature at the polymerization is also important to the size of thepores.

The upper limit of the size of the pores is determined by the size ofthe micro-organisms which are encapsulated in the gel body, said limitbeing some tenth or tenths of a μm. The lower limit of the size of thepores is determined by the molecular size of a) the components of thenutrient solution which is to diffuse into the gel body at incubation,and b) of metabolites from the micro-organism which are to diffuse outinto the nutrient solution.

The gel body can be dried or aqueous, i.a. depending on the physicalcondition of the product to be sterilized. When used for sterilizationcontrol of e.g. food stuff, a high percentage of water imparts to thegel heat transferring properties which are identical with those of mostliquid and solid food products, for which reason the heat killing effectin the gel can be directly applied to the respective product. Suitablewater-containing gels are polyacrylamide with a water content exceeding50%, calcium alginate or gelatine having more than 90% water, andpolyurethane gel likewise containing more than 90% water.

The gel body according to the invention should have a delimited physicalform, adjusted to the product subject to sterilization control and tothe process equipment, and it can have a diameter from some millimeterup to ten millimeters or so, for example, it could have the size of apea.

The micro-organism encapsulated in the gel body should be a welldefined, apathogenic micro-organism having a known and reproducibleresistance, and it can be a spore or a bacterium, a mould or yeastfungus etc. The resistance is defined by a so called D-value (DecimalReduction Value), that is, the time of action/temperature/dose whichwill give a 90% kill of the spores. For e.g. Bacillus stearothermophilusthe D-value at 121° C. will be about 1,5 minutes and for e.g. Bacillussubtilis the D-value at 160° C. will be 5-10 minutes.

Suitable micro-organisms are Bacillus subtilis, for example Niger ATCC9372 (ATCC=The American Type Culture Collection, USA) or SSI MK1(SSI=initials of the Strain Stock of the State Serum Institute, Denmark)and Bacillus stearothermophilus, for example, ATCC 7953 or NIH 7953(NIH=National Institute of Health Collection, USA). These organisms arepreferably used in the form of spores.

An important feature of the gel body according to the invention is thefact that it has to be thermally stable in various sterilizationprocesses, that is, it has to be thermally stable at the sterilizationtemperature concerned, e.g. up to 140° C. Furthermore, the mechanicalstrength of the gel body has to be such that it will endure transportthrough pumps, conduits, heat exchangers etc.

Furthermore, the gel body has to be surface sterile; surfacesterilization of the gel body is preferably carried out by the bodybeing treated with an aqueous solution of hydrogen peroxide or subjectto gas sterilization by formaldehyde or ethylene oxide.

The gel body should also be transparent; transparency allows the resultof the control to be swiftly and simply indicated thanks to the factthat, for example, a colour change at incubation can be readily observedand a possible microscopic checking of growth can be readily carriedout.

The gel body according to the invention has preferably the form of asphere having a diameter of 1-10 mm. An embodiment, particularlypreferred, of a "gel ball" according to the invention includes an innerball (a core) of e.g. polyacrylamide gel with spores of a micro-organismencapsulated therein, plus a surface sterile outer cover, e.g. of acalcium alginate gel or gelatine, such combination giving a particularlyspore-tight and surface sterile gel ball. Such a gel ball can beproduced as follows:

PRODUCTION OF A PREFERRED GEL BODY 1. Preparation of a PolyacrylamideCore.

Three stock solutions are made. 25-35 g acrylamide and 0.5-1.5 gN,N'-methylenebis-acrylamide (bis) are prepared with water to 100 ml(stock solution A). 75-125 mg ammonium persulphate is dissolved in 1 mlwater (stock solution B) and 0.1 ml TEMED(N,N,N',N'-tetramethyl-ethylenediamine) is dissolved in 1 ml water, towhich is also added varying amounts of spores, e.g. of Bacillus subtilis(stock solution C). By diluting 1-5 ml A to 15 ml and adding 20-100 μl Band 20-100 μl C a basic solution for polymerization is obtained, whichcontains about 2-8% acrylamide and 0.1-0.5% bis. In a particularembodiment, by diluting 2.5 ml A to 15 ml and adding 40 μl B and 60 μlC, a basic solution for polymerization is obtained, which contains about5% acrylamide and 0.2% bis. Now, from this solution a polyacrylamidebody is molded, which is allowed to cure at 0-50° C., preferably at25-30° C. Thereafter, the body is allowed to dwell for 5-6 min indistilled water.

2. Application of a Calcium Alginate Layer

The polyacrylamide body is dipped into a calcium alginate solutioncontaining more than 90% water, preferably more than 97% water, at roomtemperature. Thereafter, an alginate film is precipitated on the gelbody of polyacrylamide by the body being immersed in a salt solution,e.g. calcium chloride in water, at a concentration of at least 1-3%.Precipitation occurs during 1-20 hours at room temperature. The gel bodyis thereafter kept in distilled water.

3. Surface Sterilization of the Gel Body.

The surface of the gel body of polyacrylamide/calcium alginate isthereafter sterilized by the body being treated with a solution of e.g.hydrogen peroxide in a concentration of 1-35%, after which the gel iskept in distilled water.

The steps 2 and 3 above can be repeated one or more times in order thata tight and surface sterile gel ball be ensured.

In carrying out the method according to the invention the gel body orbodies are added to the product to be heat treated. Then sterilization,pasteurization or cooking takes place in a conventional manner bycarrying out, in batches or continously, a pressure or temperaturetreatment, steam sterilization, hot air sterilization, formaline orethylenoxide sterilization etc.

When treating e.g. meat broth or starch solution in batches in anautoclave, 10-100 gel bodies or gel balls are added per 1000 liters ofthe product after which the product is treated for about 20 minutes inautoclave at about 121° C. When sterilizing continuously, gel bodies areadded at desired time intervals to the product subject to continuoussterilization, or they are added in connection with restarting of acleaned process unit, after which the product is passed through aheating zone during 1-10 minutes at a temperature of up to 140° C.

After sterilization having been completed the gel bodies are separatedin a suitable manner. This can be carried out by filtering, flotation,sedimenting or the like, depending on the density of the gel body and onthe form and state of the sterilized product.

After separation of the gel bodies from the sterilized product each gelbody is aseptically transferred to a tube with a nutrient solutionsuited to the micro-organism. One way of establishing a possible growthof the spores in the gel body is by means of a colour indicator, e.g.BTB-bromothylmol blue, component of the nutrient solution. Growth, andacid produced thereby, will result in a decrease of pH, which causes thecolour of the indicator to change from blue via green to yellow. Readingtakes place within 1-3 days; a preliminary reading could perhaps be madealready after 10-15 hours. Thereby an answer is obtained more quickly incase the process has been incomplete, meaning, that the spores have notbeen killed.

The reason for this lack of effect can be attributed to a defectsterilizer or difficult sterilizing conditions. The problem has to beidentified immediately so that the necessary measures can be taken.

A possible growth of the spores can also be established visually by amicroscopic inspection of colonies of vegetative cells. This offers thepossibility to read quantatively the killing of spores which the processhas brought about.

The gel body and the method of sterilizing control according to theinvention can be applied within various fields, for example, in the foodindustry and there particularly for sterility control of continuouslyprocessed liquid products, or products containing pieces, such as peasoup, meat broth, starch solution etc., Thanks to the fact that the gelbody, which in this case contains water and has heat transferringproperties almost identical with those of the product, is subjected toexactly the same sterilizing conditions as the product, a reliable andeffective sterility control of the product is attained.

We claim:
 1. A method for carrying out biological control of asterilization process which comprises:(a) adding to a product to besterilized one or more gel bodies consisting essentially of a gel andencapsulated spores of an apathogenic micro-organism having a known andreproducible resistance, defined by the time of action/temperature/dosewhich will give a 90% kill of the spores, and further wherein said gelof said gel body consists essentially of a core of a gel, selected fromthe group consisting of polyacrylamide, gelatine, agar, calciumalginate, polyurethane and combinations thereof, and one or more layersof a gel of calcium alginate or gelatine applied thereon, and furtherwherein the gel body has a porous structure, preventing the encapsulatedspores from diffusing out but allowing a nutrient solution adjusted tothe micro-organism to diffuse in and metabolites produced to diffuseout; is thermally and mechanically stable to sterilization,pasteurization and cooking; is surface sterile; and is transparent inorder to allow observation of possible growth of the micro-organism whenincubated in a nutrient solution; (b) exposing the product containingsaid one or more gel bodies to sterilizing conditions at elevatedtemperatures; (c) separating the gel body or bodies from the sterilizedproduct, after sterilization has been completed, in an aseptic manner;and (d) incubating the gel body or bodies of step (c) in a nutrientsolution in order to determine possible growth of the micro-organism. 2.A gel body having a delimited physical form wherein the gel bodyconsists essentially of a gel and encapsulated spores of an apathogenicmicro-organism having a known and reproducible resistance to heat,defined by the time of action/temperature/dose which will give a 90%kill of spores, and further wherein said gel of said gel body consistsessentially of a core of a gel selected from the group consisting ofpolyacrylamide, gelatine, agar, calcium alginate, polyurethane andcombinations thereof, and one or more layers of a gel of calciumalginate or gelatine applied thereon; and further wherein the gel bodyhas a porous structure, preventing the encapsulated spores fromdiffusing out but allowing a nutrient solution adjusted to themicro-organism to diffuse in and metabolites produced to diffuse out; isthermally and mechanically stable to sterilization, pasteurization andcooking; is surface sterile; and is transparent in order to allowobservation of possible growth of the micro-organism when incubated in anutrient solution.
 3. The gel body as in claim 2, wherein theencapsulated spores of an apathogenic micro-organism are temperaturespecific.
 4. The gel body as in claim 3, wherein the temperaturespecific encapsulated spores of an apathogenic micro-organism areselected from the group consisting of Bacillus subtilis and Bacillusstearothermophilus.
 5. The gel body as in claim 2, wherein the surfacehas been sterilized by treating the surface with hydrogen peroxidesolution and then placing the gel body in distilled water.
 6. A methodfor carrying out biological control of a sterilization process whichcomprises:(a) adding to a product to be sterilized one or more gelbodies consisting essentially of a gel and encapsulated spores of anapathogenic micro-organism having a known and reproducible resistance,defined by the time of action/temperature/dose which will give a 90%kill of the spores, and further wherein said gel of said gel bodyconsists essentially of a core of a gel selected from the groupconsisting of polyacrylamide, gelatine, agar, calcium alginate,polyurethane and combinations thereof, and one or more layers of a gelof calcium alginate or gelatine applied thereon; and further wherein thegel body contains water; has a porous structure, preventing theencapsulated spores from diffusing out but allowing a nutrient solutionadjusted to the micro-organism to diffuse in and metabolites produced todiffuse out; is thermally and mechanically stable to sterilization,pasteurization and cooking; is surface sterile; and is transparent inorder to allow observation of possible growth of the micro-organism whenincubated in a nutrient solution; (b) exposing the product containingsaid one or more gel bodies to sterilizing conditions at elevatedtemperatures; (c) separating the gel body or bodies from the sterilizedproduct, after sterilization has been completed, in an aseptic manner;and (d) incubating the gel body or bodies of step (c) in a nutrientsolution in order to determine possible growth of the micro-organism. 7.A gel body having a delimited physical form wherein the gel bodyconsists essentially of a gel and encapsulated spores of an apathogenicmicro-organism having a known and reproducible resistance to heat,defined by the time of action/temperature/dose which will give a 90%kill of spores, and further wherein said gel of said gel body consistsessentially of a core of a gel selected from the group consisting ofpolyacrylamide, gelatine, agar, calcium alginate, polyurethane andcombinations thereof, and one or more layers of a gel of calciumalginate or gelatine applied thereon; and further wherein the gel bodycontains water; has a porous structure, preventing the encapsulatedspores from diffusing out but allowing a nutrient solution adjusted tothe micro-organism to diffuse in and metabolites produced to diffuseout; is thermally and mechanically stable to sterilization,pasteurization and cooking; is surface sterile; and is transparent inorder to allow observation of possible growth of the micro-organism whenincubated in a nutrient solution.
 8. The gel body as in claim 7, whereinthe encapsulated spores of an apathogenic micro-organism are temperaturespecific.
 9. The gel body as in claim 8, wherein the temperaturespecific encapsulated spores of an apathogenic micro-organism areselected from the group consisting of Bacillus subtilis and Bacillusstearothermophilus.
 10. The gel body as in claim 7, wherein the surfacehas been sterilized by treating the surface with hydrogen peroxidesolution and then placing the gel body in distilled water.